A series of 9-aminoacridine spin labels has been synthesized (see Project No. ZO1 HL 02517-01 PB) and their electron spin resonance (ESR) spectra recorded in the presence of DNA, poly dA-dT and poly dG-dC. A comparison of the maximum anisotropic hyperfine splittings (A2) for 4-(6-chloro-2-methoxy-9-acridinyl)-amino)-2,2,6,6-tetramethyl-1-piperidinyloxy (I) and 4-(9-acridylamino)-2,2,6,6-tetramethyl-1-piperidinyloxy (II) bound to DNA, poly dA-dT and poly dG-dC provided direct evidence that II preferred dA-dT base-pairs while I showed no base preference. The methiodide (III) and ethiodide (IV) salts of II also seemed to bind preferentially to dA-dT base-pairs. Polydeoxyridbonucleotide complexes with label II all exhibited higher melting temperatures (Tm) than complexes with lable I. These results suggest the double helix is stabilized by the interaction of the acridine ring with the phosphate backbone so that the substitution of a bulky group on the ring will decrease binding. The ESR spectra of I and II in RNA showed the existence of two distinct species. Comparison of the ESR spectra of I and II in RNA with those in poly A-poly U, poly G-poly C, poly A, poly U, poly C and poly G indicates that the two species were due to single- and double-stranded complexes in RNA. Hence, these labels may serve as useful tools in studying base-pairs specificity during intercalation as well as useful probes for single- and double-stranded regions on nucleic acids.